Esterases belong to the family of nonspecific enzymes that catalyze the hydrolysis of esters. Human esterase D (ESD) is one member of the esterase family distinguishable by its electrophoretic mobility and its relative specificity for methylumbelliferyl esters as substrate. Human ESD is the dimeric enzyme in that it displays several phenotypes as a result of the expression of codominant autosomal alleles, primarily allele ESD 1 and ESD 2. Such polymorphic nature of human ESD has been shown to be available marker in studies of population genetics. Am. Hum. Genet., 39: 1-20 (1975).
The activity of ESD enzyme depends on the normal function of the ESD gene. Consequently, absence, complete or partial inactivation, deletion of one ESD allele, mutation or other alterations in ESD sequences will result in decreasing of ESD activity. For example, the tissues of individuals with a deletion of one chromosome 13 show only 50% of the ESD activity of that found in the healthy individuals possessing a normal set of two chromosomes 13. Science, 219:973-975 (1983).
The genetic locus of ESD was mapped to the chromosome 13q14:11 region by correlating the loss of enzyme activity with deletions on chromosome 13. Science, 208:1042-1044 (1980). The regional assignment of ESD to 13q14:11 region coincides with the location of the retinoblastoma (RB) gene, shown to be involved in the tumorigenesis of retinoblastoma. Am. J. Dis. Child., 132:161-163 (1978); Science, 219:973-975; Science, 213:1501-1503 (1981). The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14:11 is required for tumorigenesis.
By examining levels of esterase D mapping to 13q14:11, it was previously inferred that one chromosome 13 in the somatic cells of the retinoblastoma patient contained a submicroscopic deletion of the RB and esterase D loci and that this chromosome was retained in her tumor, while the normal chromosome 13 was lost. Cancer Gen. Cytogen, 6:213-221 (1982).
Retinoblastoma is a malignant tumor of the sensory layer of the retina. The neoplastic tumor is composed of primitive retinal cells, occurring either bilaterally or unilaterally, usually before the third year of life. Retinoblastomas are characterized by small round cells with deeply staining nuclei, and elongated cells forming rosettes. They cause death by usually local invasion, especially along the optic nerves.
The molecular mechanism of the formation of this tumor is unknown. Absence or inactivation of the RB gene is believed to be the primary cause of this inheritable, childhood cancer. Science, 213:1501-1503 (1981); Science, 223: 1028-1033 (1984); Proc. Natl. Acad. Sci., 68:820-823 (1971); Nature, 305:779-784 (1980). Since little of the RB gene structure or function is known, its cloning proved to be difficult. Discover, March:95-96 (1987). The difficulties with cloning of the RB gene were similar to that encountered in cloning the muscular dystrophy or the cystic fibrosis genes. Nature, 316:842-845 (1985), Science, 230:1054-1057 (1985).
The latest reports indicate that the RB gene has a regulatory function and that its presence and normal function prevents the development of the retinoblastoma. On the other hand, absence, malfunctioning or inactivation of the RB gene causes the development of, or genetical predisposition and susceptibility to, the retinoblastoma and is believed to be the primary cause for both hereditary and acquired retinoblastoma, and for the secondary malignancies often recurring in retinoblastoma patients such as osteosarcoma, and fibrosarcoma.
Therefore, to find the way how to determine the genetic predisposition in fetus or the susceptibility to acquire retinoblastoma in later age is of utmost importance for early diagnosis and/or possible treatment through a genetic manipulation.
The localization of the ESD and RB genes in the same chromosomal region provides an advantageous approach for evaluation of the RB gene functioning, for discovery of RB chromosomal patterns and for cloning of the RB gene using the ESD as the starting point. Science, 235:1394-1399 (1987). The tight linkage between these two genes allows the ESD gene to serve as a crucial marker in elucidating the behavior of the RB gene. Science, 219:973-975 (1982); and Nature, 304:451-453 (1983).
Thus, the identified and cloned ESD cDNA would be advantageous in the identification of the RB gene location which in turn would allow RB diagnosis and treatment.
Besides the RB gene, the defective gene in Wilson's disease has been found to be located in the same chromosomal region 13q14:11, and thus was found to be linked to the ESD gene. Proc. Natl. Acad. Sci., 82:1819-1821 (1985). Wilson's disease, also known as hepatolenticular degeneration, is an hereditary disease of ceruloplasmin formation transmitted as an autosomal recessive. It is characterized by gross reduction in the incorporation of copper in ceruloplasmin resulting in decreased serum ceruloplasmin and copper values, and increased excretion of copper in the urine. The close linkage found between Wilson's disease locus and the ESD gene, which can serve as the polymorphic marker for Wilson's disease, has the profound implications both for investigation of the primary gene defect and for clinical use.
The identified and cloned ESD cDNA thus would provide a valuable marker in the identification of the Wilson's disease gene and would lead, eventually, to diagnosis and treatment of this inheritable genetic disease.
In addition to serving as a genetic marker for retinoblastoma and Wilson's disease, the ESD may play a role in detoxification. It has been recently observed that the ESD protein is distributed at the highest level in liver and kidney and that it is inducible by phenobarbital, but not phorbol myristate ester treatment. Proc. Nat. Acad. Sci., 83:6790-6794 (1986).
Although the esterase D has been found in most tissues, not much is known about its structure and function. A protocol for partial purification of this enzyme has been described previously but the purity achieved by the procedure was only 30%--40%. Am. J. Hum. Genet., 30,14-18 (1978). Thus, it remains difficult to obtain a sufficient amount of the purified, homogeneous enzyme to generate specific anti-esterase D antibodies.
It would be, therefore, valuable to obtain a highly purified form of human ESD and to develop a method of preparing such highly purified human ESD protein. Further, it would be desirable to prepare specific anti-esterase D antibodies, and to isolate a human ESD cDNA clone, to identify the human ESD cDNA and ESD gene nucleotide sequence and human ESD amino acid sequence.
It is therefore one object of this invention to provide a purified human ESD and to determine its amino acid sequence.
It is another object of the present invention to provide a process for purifying human ESD.
It is yet another object of this invention to provide a specific polyclonal antibody against a purified human ESD.
It is still another object of the invention to provide the human ESD cDNA and to determine its nucleotide sequence.
It is still another object of the invention to clone ESD cDNA and to isolate and construct the ESD cDNA probes.
It is a further object of the present invention to provide a human ESD cDNA radioactive probe which can be utilized as a genetic marker for retinoblastoma and Wilson's disease.
It is still a further object of this invention to identify and isolate human ESD gene and to determine its nucleotide sequence.